Evaluation of ex vivo drug combination optimization platform in recurrent high grade astrocytic glioma: An interventional, non-randomized, open-label trial protocol

Introduction High grade astrocytic glioma (HGG) is a lethal solid malignancy with high recurrence rates and limited survival. While several cytotoxic agents have demonstrated efficacy against HGG, drug sensitivity testing platforms to aid in therapy selection are lacking. Patient-derived organoids (PDOs) have been shown to faithfully preserve the biological characteristics of several cancer types including HGG, and coupled with the experimental-analytical hybrid platform Quadratic Phenotypic Optimization Platform (QPOP) which evaluates therapeutic sensitivity at a patient-specific level, may aid as a tool for personalized medical decisions to improve treatment outcomes for HGG patients. Methods This is an interventional, non-randomized, open-label study, which aims to enroll 10 patients who will receive QPOP-guided chemotherapy at the time of first HGG recurrence following progression on standard first-line therapy. At the initial presentation of HGG, tumor will be harvested for primary PDO generation during the first biopsy/surgery. At the point of tumor recurrence, patients will be enrolled onto the main study to receive systemic therapy as second-line treatment. Subjects who undergo surgery at the time of recurrence will have a second harvest of tissue for PDO generation. Established PDOs will be subject to QPOP analyses to determine their therapeutic sensitivities to specific panels of drugs. A QPOP-guided treatment selection algorithm will then be used to select the most appropriate drug combination. The primary endpoint of the study is six-month progression-free survival. The secondary endpoints include twelve-month overall survival, RANO criteria and toxicities. In our radiological biomarker sub-study, we plan to evaluate novel radiopharmaceutical-based neuroimaging in determining blood-brain barrier permeability and to assess in vivo drug effects on tumor vasculature over time. Trial registration This trial was registered on 8th September 2022 with ClinicalTrials.gov Identifier: NCT05532397.


Title:
Evaluation of ex vivo drug combination optimization platform in recurrent high grade astrocytic glioma

Objectives:
Our central hypothesis is that PDOs mimic the biological characteristics of high grade astrocytic gliomas and serve as an ideal platform for the evaluation of drug sensitivities, accurately reflecting the patient's therapeutic response to the drugs.This will enable us to facilitate therapeutic decisionmaking and personalised anti-cancer therapy.In this study, we will specifically evaluate drug sensitivities/ resistance of high grade astrocytic glioma organoids before and after standard-of-care first line therapy, and match these with treatment outcomes, with the objective of determining if QPOP analysis can effectively predict therapeutic sensitivities as well as determine the optimal treatment at the time of relapse.High grade astrocytic glioma is an ideal tumor type to test our hypothesis because there is currently no standard-of-care second line systemic therapy for this disease.In addition, the blood-brain barrier (BBB) poses significant challenges to the delivery of systemic therapy to brain tumors.This study presents a unique opportunity to examine the relationship between individualized tumoral therapeutic sensitivity, BBB permeability and patient outcomes.We aim to evaluate how BBB permeability evolves over time from first diagnosis to tumor recurrence using Gallium-68 NOTA-Evans Blue (Ga68-NEB) Positron Emission Tomography/ Magnetic Resonance Imaging (PET/MRI) and Dynamic Contrast Enhanced (DCE)-MRI imaging, and will correlate imaging biomarkers with QPOP-derived therapeutic sensitivities, clinical and radiological response as well as patient outcomes.

Trial Design:
This is an interventional, non-randomized, single site study.In the pilot phase of our study, we will aim to enrol 10 patients who will receive QPOP-guided systemic therapy at the time of first high grade astrocytic glioma recurrence.Subjects will be replaced accordingly if their QPOP analysis is unsuccessful or its results do not lead to therapeutic decision making.

Pre-screening phase:
Patients from NUH and NTFGH (involve in only Pre-screening phase) will be approached for prescreening consent at the time of first suspected diagnosis of a high grade astrocytic glioma and tumor will be harvested at the time of initial surgery/ biopsy for the generation of PDOs.Once the histological diagnosis of high grade astrocytic glioma is confirmed and the patient is planned for adjuvant temozolomide and radiotherapy, our prescreening cohort will undergo baseline standard MRI plus Ga68-NEB PET/MRI and DCE-MRI imaging prior to chemoradiotherapy (Radiological timepoint 1).If patients are admitted to the ward, they will not be sent for the Ga68-NEB PET/MRI and DCE-MRI imaging.These scans should be arranged as outpatient scans only after they are discharged from the ward.In addition, tumor may be harvested for any subsequent procedure that patient might require for brain tumor treatment.Study imaging and further PDO generation will not take place if the patient does not meet the histological criteria or will not be receiving standard adjuvant temozolomide/ radiotherapy.It is estimated that we will need to prescreen approximately 20 patients in order to achieve our objective of enrolling 10 recurrent high grade glioma patients treated with QPOP-guided systemic therapy.
Patients will then undergo standard-of-care treatment for high grade astrocytic glioma with adjuvant concurrent temozolomide and radiotherapy (TMZ/RT) 1 , and will be evaluated by the primary oncologist routinely with clinical examination, laboratory tests and regular neuro-imaging (performed as standard clinical care and not part of the study).High grade astrocytic glioma organoids will be generated from resected tumour samples and QPOP analyses of the treatmentnaïve high grade astrocytic glioma organoids will be performed (QPOP 1).To model TMZ/RTresistant cells in the setting of recurrent high grade astrocytic glioma, these treatment-naïve organoids will be subjected to chronic exposure of temozolomide, and ionizing radiation.QPOP analyses will be performed on these resistant high grade astrocytic glioma organoids to identify specific drug combinations to guide clinical management at the time of first relapse (QPOP 2).

Main study:
At the time of documented tumor recurrence, eligible patients will be reassessed for suitability to participate in the main study and approached for their informed consent to enter this phase of the study.At this juncture, a small proportion of patients will be deemed suitable for a second operation.If this is the case, high grade astrocytic glioma organoids will also be generated from the recurrent high grade astrocytic glioma tumour and subject to QPOP analyses (QPOP 3).During the main study, patients will receive QPOP-guided systemic therapy for the treatment of their relapsed high grade glioma and will be assessed regularly for safety and efficacy of this therapy.In addition, patients will undergo standard MRI plus investigational Ga68-NEB PET/MRI and DCE-MRI imaging prior to and 8 weeks (+/-1 week) after QPOP-guided systemic therapy (Radiological timepoints 2 and 3, respectively).If patients are admitted to the ward, they will not be sent for the Ga68-NEB PET/MRI and DCE-MRI imaging.These scans should be arranged as outpatient scans only after they are discharged from the ward.Subsequent to this, radiological assessment of their disease will revert to standard clinical protocols with routine standard MRI imaging.

Choice of dosing regimen
We will have two in vitro drug screening panels.][3][4][5][6][7][8][9][10][11][12][13] All of these drugs have been determined to have sufficient penetration across the blood-brain barrier and have known adverse event profiles.QPOP will be used to derive top-ranking drug combinations, which will then be validated in vitro.The "experimental panel" (Panel 2) comprises anti-cancer agents which have promising activity in high grade astrocytic glioma but are not yet routinely used in high grade astrocytic glioma patients: Geftinib, osimertinib, vemurafenib, everolimus, sunitnib, regorafenib, selinexor, marizomib, abemaciclib, ivosidenib, olaparib, metformin.The results of this panel will not be used in the care of our enrolled patients; instead they are hypothesis-generating and intended for us in designing future trials.
The drug combinations generated by QPOP can be divided into 3 categories: 1) combination regimens with published data in the setting of gliomas (category 1); 2) combination regimens where there is published data on intracranial activity and anti-glioma effect of the individual agents (either as monotherapy or in combination with other agents), and where there is published safety data on the combination in non-glioma settings (category 2); and 3) novel drug combinations that have not been assessed for clinical efficacy (category 3).
We will apply the following guidelines for the selection of drug regimen in individual patients: 1.If QPOP analysis results of QPOP 1, 2 and 3 are all available at the time the patient is due to start systemic therapy for the treatment of recurrent high grade astrocytic glioma (i.e., 4-6 weeks after surgery), they will be used in the following order of preference: QPOP 3> QPOP 2> QPOP 1, provided other eligibility criteria for the use of that particular combination's results are met.
2. We will make every effort to use category 1 combinations, but will consider category 2 combinations should the QPOP analysis not generate any category 1 combinations.Decisions to choose category 2 combinations vs defaulting to monotherapy (see point 3) will be discussed with the study team.Doses of drug combinations used should be as close as possible to those used in the published literature.We will not use novel drug combinations (category 3) that have not previously been assessed for clinical safety.
3. If the QPOP combination therapy results do not fall within the criteria for usage, monotherapy may be considered.Temozolomide monotherapy will not be encouraged unless strongly supported by QPOP 2 or 3 results, in which case, a dose schedule different from first-line therapy should be used.
4. Bevacizumab is an FDA approved drug for the treatment of recurrent high grade astrocytic glioma (Avastin (bevacizumab) injection, Genetech, Inc, December 2017), and may be used in combination with lomustine 11 , temozolomide 10,11 , irinotecan 10 , carboplatin 7 and etoposide 7 for the treatment of high grade astrocytic glioma.However, bevacizumab is not included in the drug screening panel because our current PDO models lacks vasculature, hence the functional testing of antiangiogenic drugs is irrelevant.In order to be relevant to real-world practice, the study will allow the addition of bevacizumab to any of the above agents, provided that investigators adhere as closely as possible to the regimens in the referenced studies.The addition of bevacizumab will not be permitted if the QPOP results suggest an effective combination where there is no published data for the addition of bevacizumab to that particular treatment combination.Deviations from existing data may be permitted only with the agreement of the study team.
The study team will meet to review the QPOP analysis results to agree on the validity of the data and determine the recommended treatment at the time of relapse for each patient on the study.As an additional safety measure, the study team will also conduct a combined review for each patient at the time of their first post-treatment radiologic assessment, to ensure the appropriateness of therapy based on the efficacy and toxicity of the treatment selected.

Subject Population:
Male and female subjects aged 21 years and above with suspected high grade astrocytic glioma planned for surgery/ biopsy followed by adjuvant chemoradiotherapy will be invited to participate in the pre-screening study.Subjects will only be enrolled in the main study if they had pathologically confirmed high grade astrocytic glioma, and received adjuvant treatment comprising standard-of-care therapy with surgery/biopsy followed by temozolomide and radiotherapy.In addition, they are required to have had sufficient tumor tissue available for PDO generation at baseline.We aim to enrol 10 patients who will receive QPOP-guided chemotherapy at the time of first high grade astrocytic glioma recurrence.In order to achieve this, we expect to pre-screen approximately 20 subjects.Subjects will be replaced accordingly if their QPOP analysis is unsuccessful or its results do not lead to therapeutic decision making.These subjects will not be included in our study endpoint analysis.This study is expected to take 18-24 months to complete.
Written informed consent for entry into the study will be obtained prior to any study specific procedure.All eligibility criteria and consent forms will be checked before treatment is initiated.

Inclusion Criteria
Subjects may be included in the study only if they meet all of the following criteria: Pre-screening: 1) Patients 21 years of age or older, with ECOG performance status 0 to 2, and with a life expectancy of more than 3 months with suspected high grade astrocytic glioma, fit for treatment comprising standard-of-care therapy with adjuvant temozolomide and radiotherapy if the diagnosis of high grade astrocytoma is pathologically confirmed.2) Signed informed consent obtained before any study specific procedure.Subjects must be able to understand and be willing to sign the written informed consent.

All subsequent criteria apply to the main study only:
1) Patients 21 years of age or older, with ECOG performance status 0 to 2, and life expectancy of more than 3 months with pathologically confirmed high grade astrocytic glioma, having undergone first-line standard-of-care therapy with surgery/biopsy followed by temozolomide and radiotherapy.Subjects with truncated adjuvant chemoradiotherapy may be enrolled at the Principal Investigator's discretion.2) Documented tumor progression based on standard clinical, radiological or histological criteria, and deemed suitable for second line systemic therapy.3) Sufficient tumor tissue available for PDO generation at baseline and at least one available or pending QPOP result.4) Adequate organ function as defined by: 1. Bone marrow function i. Haemoglobin ≥ 9g/dl ii.Absolute neutrophil count (ANC) ≥ 1.5 x 10 9 /L iii.Platelet count ≥ 100 x 10 9 /L. 2. Liver function i. Bilirubin < 2.5x upper limit of normal (ULN) ii.Alanine transaminase (ALT) and aspartate transaminase (AST) < 2.5x ULN or < 5x ULN if liver metastases are present iii.Prothrombin time (PT) within the normal range for the institution.3. Renal function i. Plasma creatinine <1.5x institutional ULN 5) Capable of swallowing tablets.6) Recovery from any previous drug-or procedure-related toxicity to National Cancer Institute Common Terminology Criteria for Adverse Events (NCI-CTCAE) version 5.0 Grade 0 or 1 (except alopecia), or to baseline preceding the prior treatment.

Exclusion Criteria (both pre-screening/ main study)
1) Chemotherapy, radiotherapy, surgery, immunotherapy or other therapy within 2 weeks of study entry.
2) Pregnancy or breastfeeding at the point where systemic anti-cancer therapy is initiated.Women of childbearing potential must have a negative pregnancy test at the point where systemic anti-cancer therapy is initiated.Women of childbearing potential and men, must agree to use adequate contraception (barrier method of birth control) while on anti-cancer treatment and until at least 3 months after the last study drug administration.3) Concurrent cancer which is distinct in primary site or histology from the cancer being evaluated in this study EXCEPT cervical carcinoma in situ, treated basal cell carcinoma, superficial bladder tumours (Ta, Tis & T1) or any cancer curatively treated less than 5 years prior to study entry.4) Patients with leptomeningeal dissemination of disease and/or pure spinal high grade gliomas will be excluded.5) Kidney disease which would clinically disqualify the subject from serial MRI scans with gadolinium contrast.6) Previous history of allergy to MRI contrast agents.

Study Assessments
Clinical (within 2 weeks of study enrolment and at the start of each cycle of treatment): • Medical history and physical examination, evaluation of performance status (ECOG) • Adverse event assessment (CTCAE v 5.0) Radiologic (pre-screening phase: prior to chemoradiotherapy; main study: prior to and 8 weeks (+/-1 week) after QPOP-guided systemic therapy): • Standard MRI brain +/-perfusion as indicated Laboratory (within 2 weeks of study enrolment and at the start of each cycle of treatment, +/-3 days window period): • Laboratory parameters including haematology (FBC), chemistries, others as clinically indicated

Final Study Visit:
Clinical assessment within 30 days from the time of study discontinuation/completion of study treatment.

Gallium-68 NOTA-Evans Blue (Ga68-NEB) Positron Emission Tomography/ Magnetic Resonance Imaging (PET/MRI) and Dynamic Contrast Enhanced (DCE)-MRI imaging
Standard MRI brain with or without perfusion sequences are typically performed at baseline and after therapy in order to evaluate radiological response.In this study, we plan to add Ga68-NEB (Gallium-68 NOTA-Evans Blue) PET imaging and DCE-MRI imaging to examine BBB permeability and assess in-vivo drug effects on tumor vasculature over time.These studies are planned at 3 timepoints: 1) During the pre-screening phase, following confirmation of histological diagnosis and prior to chemoradiotherapy and 2) during the main study, prior to and 8 weeks (+/-1 week) after QPOP-guided systemic therapy.Radiological biomarkers will be correlated with radiological response based on follow up with routine MRI and clinical outcomes as well as therapeutic sensitivities.

Radiomics
MRIs obtained at initial diagnosis and subsequent scans will be subjected to radiomic feature extraction, and correlated with spatially annotated histopathological biopsies opportunistically obtained as part of standard-of-care surgical resection both at initial diagnosis (chemotherapy-and radiotherapy-naïve) and at recurrence.Annotation of biopsy site would be done on T1-stereotaxy and pathology images would be correlated with spatial radiomic features and clustering.In addition, part of the biopsy will be used to generate high grade astrocytic glioma organoids, and tested for chemosensitivity towards both temozolomide and the optimal drug combination the participant will be treated with.These spatial properties of the high grade astrocytic glioma biopsies will be used for model building for prediction of post-chemotherapy recurrence.

b) Histopathology
Paraffin-embedded blocks of high grade astrocytic glioma tumour will be stained to assess DNA damage response (ɣH2AX, RAD51), growth factor receptors (EGFR, PDGFR) and hypoxia markers (HIF1A) in relation to blood vessels and axons.Molecular analyses of TP53, ATRX, IDH1, IDH2, BRAF, and EGFRvIII mutations, MGMT methylation status, and 1p19q co-deletion would be performed as part of routine care.

c) Plasma biomarkers
Approximately Fifteen to Eighteen millilitres of blood will be collected together with routine blood tests for plasma biomarkers including circulating tumor DNA, proteomics and DNA methylation at these time points.These will be stored in the NUH tissue repository for future analysis.
• Prescreening -At first MRI scan before chemoradiotherapy • Main study -At baseline and 8-week post QPOP guided chemotherapy

Outcomes Measures and Statistical Plan
This is an interventional, non-randomized, single site study.The primary purpose of this study is not hypothesis testing, but to assess the feasibility of QPOP-guided therapy for recurrent high grade astrocytic glioma to be used in a larger scale study.Therefore, this study does not have a formal sample size, but rather, a set benchmarks to determine feasibility.We plan to enrol a total of 10 patients who receive QPOP-guided chemotherapy at the time of first high grade astrocytic glioma recurrence.We expect this to be sufficient for determining the feasibility of a phase II trial in which there will be formal sample size calculations for the achievement of PFS6 > 30%. 35In addition, a phase II study will only be considered feasible if there is successful organoid generation and QPOP analysis in > 50% of samples collected.In this study, subjects will be replaced accordingly if their QPOP analysis is unsuccessful or its results do not lead to therapeutic decision making.

Efficacy Endpoints
Primary clinical endpoint: i. Six-month progression-free survival (PFS 6).PFS is defined as the time from the start of study treatment to documented progression of disease or death; PFS6 refers to the percentage of patients who are alive and free of high grade astrocytic glioma progression at 6 months.

Secondary clinical endpoints:
ii. Radiological response assessments at follow up MRI.The determination of radiographic response is as per the Response Assessment in Neuro-Oncology (RANO) criteria. 14iii.Twelve-month overall survival (OS12).OS is defined as the length of time from the start of study treatment, that patients diagnosed with the disease are still alive.OS12 refers to the percentage of patients who are alive at 12 months.iv.Haematological and non-haematological toxicities.As defined by National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTCAE) v5.0

BACKGROUND
High grade astrocytic glioma is one of the most common and aggressive adult brain malignancies with limited therapeutic options.Despite a combination of advanced maximal surgical resection, radiation, and chemotherapy treatment, the mean survival of high grade astrocytic glioma patients remains at about 12 months.In addition, almost all high grade astrocytic glioma patients inevitably develop tumor recurrence and there is no standard treatment for recurrent high grade astrocytic glioma patients, making high grade astrocytic glioma one of the hardest challenges in cancer precision therapy.
Therefore, developing and evaluating therapeutic platforms that can provide clinicians with additional therapeutic sensitivity information at the patient-specific level may improve therapeutic outcomes in high grade astrocytic glioma patients, particularly in the recurrence setting.Quadratic Phenotypic Optimization Platform (QPOP) determines the ranking of drug combination response in a biological model system such as patient-derived organoids, and has been successfully applied in haematological malignancies such as multiple myeloma and lymphomas.Due to the challenges in reliably generating clinically meaningful patient-derived organoids in solid tumors, QPOP has not been applied in solid tumors.Recently, we have successfully cultured several high grade astrocytic glioma organoids in vitro.This proposal aims to test the feasibility of QPOP application in high grade astrocytic glioma, by first establishing clinically relevant high grade astrocytic glioma organoid models, followed by the utility of QPOP-derived drug combinations in high grade astrocytic glioma.We hypothesize that optimal QPOP-derived drug combinations from clinically relevant high grade astrocytic glioma organoids improve outcomes of recurrent high grade astrocytic glioma patients.Specifically, we will generate unique matched pair of high grade astrocytic glioma organoids with drug-and radio-resistance to model the inevitable tumor recurrence in high grade astrocytic glioma to provide clinically meaningful patient-derived models.Moving forward, we will determine the drug sensitivity of QPOP-derived drug combinations for high grade astrocytic glioma patients in drug-and radio-resistant high grade astrocytic glioma organoids as well as high grade astrocytic glioma organoids at recurrence if the same patient undergoes repeat surgery.Our study will identify and validate QPOP-guided drug combinations as a second line treatment for recurrent high grade astrocytic glioma patients.Our work will justify the application of QPOP for personalized cancer therapy in high grade astrocytic glioma patients.

Clinical Significance and Rationale for the Study
High grade astrocytic glioma are among the most devastating human adult solid malignancies.Despite advanced surgical intervention with adjuvant radiation and chemotherapy, high grade astrocytic glioma patients typically have a mean survival period of 15 months following diagnosis. 15n addition, high grade astrocytic glioma tumors demonstrate vast intra-and inter-tumor heterogeneity that confer treatment resistance which eventually result in tumor recurrence in more than 90% of all high grade astrocytic glioma patients. 16,17Currently, there is no standard treatment for recurrent high grade astrocytic glioma and the option of repeat surgical intervention is limited to about 25% of all recurrent patients. 18Clearly, there is an urgent need to develop better therapeutic strategies to improve patient outcome.One of the major obstacles for therapeutic development is the lack of clinically relevant models.At present, U87MG, U251, and T98G are some of the most commonly used two-dimensional (2D) cell research models due to their ease of access and growth.However, 2D cell cultures present limitations due to the lack of tumor heterogeneity and cellcell/matrix interactions that are present in primary patient tumor tissues. 19Despite the establishment of the National Cancer Institute 60 Panel (NCI-60) cell lines in an attempt to capture the heterogeneous nature of nine different cancer types, these 2D cell cultures are still unable to comprehensively represent the diversity which cancer patients exhibit, and is currently being phased out. 20Studies comparing the molecular profiles of cell lines with primary tumors have reported that conventional cell lines are unable to represent all cancer subtypes. 21Recently, patient-derived organoids (PDOs) have been shown to faithfully preserve and maintain heterogeneity of several primary cancer types including high grade astrocytic glioma and are being used to evaluate drug sensitivity and their associated genomic profiles. 22,23PDOs have been shown to retain the molecular profiles of the parental tumors and can be effectively used to investigate drug responses and mechanisms ex vivo. 24Thus, human PDOs represent more clinically relevant models compared to conventional cell line-based models, with the ability to recapitulate disease heterogeneity for more accurate studies of drug responses.Identifying the most suitable patient-specific drug combinations remains a challenge due to the complex molecular networks that contribute to feedback mechanisms of drug resistance and compensatory oncogenic drivers that limit efficacy of targeted inhibitors.
Current biomarker-based approaches in predicting drug sensitivity and clinical outcome are appropriate for select subpopulations and specific drugs but are not broadly applicable. 25These approaches are largely limited to monotherapy and cannot identify patient-specific drug combinations, nor do they consider other genomic alterations that are unaccounted for by the specific biomarkers used.

Preclinical Data on the Application of QPOP to Other Malignancies.
To address this critical deficit in combination therapy, we have developed an experimental-analytical hybrid platform, Quadratic Phenotypic Optimization Platform (QPOP) that can rank potential drug combination response in biological model systems.We initially applied QPOP towards identifying novel drug combinations against bortezomib-resistant multiple myeloma. 26As we have improved the efficiency of the QPOP platform and applied QPOP towards primary patient samples and patientderived organoids, it has become clear that QPOP may be useful as a clinical decision support platform that may be able to suggest clinically actionable and suitable patient-specific drug combinations derived from drug sensitivity tests using patient-derived materials.Our previous work in hematological malignancies suggest that drug combination rankings and drug sensitivity predictions by QPOP from ex vivo drug combination tests on primary patient samples can accurately identify patient-specific therapeutic options (Figure 1).For instance, QPOP was applied towards a hepatosplenic gamma-delta T-cell lymphoma (HSTCL) patient who experienced continual disease progression despite 6 lines of treatment.Following QPOP analysis of primary patient tumor cells, QPOP identified an effective combination of bortezomib/panobinostat that had previously been clinically trialed in T-cell lymphoma patients. 27Treatment using QPOP-derived therapy resulted in complete and durable response. 28Patient-specificity of this approach is highlighted in that QPOP did not recommend the same combination for every T-cell lymphoma patient sample, but rather identified a variety of combinations and therapeutic options from within a 12-drug set.
2.3 Preclinical Data on the Development of Patient Derived Organoids (PDOs) in high grade astrocytic glioma Three-dimensional (3D) organoid culture systems have been shown to capture the molecular and phenotypic features of the original parental tumors in various organs, including high grade astrocytic glioma.We have successfully cultured several high grade astrocytic glioma high grade astrocytic glioma high grade astrocytic glioma organoids in vitro and measured their growth and stemness characteristics.Importantly, we showed that high grade astrocytic glioma organoids contain a mix of both stem and non-stem cells (Figure 2), demonstrating the ability to retain the stemness and cellular heterogeneity of parental tumors.This is important as the recapitulation of complex cancer phenotypes provide more accurate predictions of the therapeutic potential of new treatments.
Using this methodology, we will extend the QPOP application to high grade astrocytic glioma patients and expect to yield important information regarding feasibility of QPOP as a clinical decision support platform as well as the diversity, frequency, and identity of patient-specific drug combinations across a range of brain tumor patients.

Hypothesis
Our central hypothesis is that PDOs mimic the biological characteristics of high grade astrocytic glioma s and serve as an ideal platform for the evaluation of drug sensitivities, accurately reflecting the patient's therapeutic response to the drugs.This will enable us to facilitate therapeutic decisionmaking and personalised anti-cancer therapy.In this study, we will specifically evaluate drug sensitivities/ resistance of high grade astrocytic glioma organoids before and after standard-of-care first line therapy, with the objective of determining if QPOP analysis can effectively predict therapeutic sensitivities at the time of relapse.high grade astrocytic glioma is an ideal tumor type to test our hypothesis because there is currently no standard-of-care second line systemic therapy for this disease.
In addition, the blood-brain barrier (BBB) poses significant challenges to the delivery of systemic therapy to brain tumors.This study presents a unique opportunity to examine the relationship between individualized tumoral therapeutic sensitivity, BBB permeability and patient outcomes.We aim to evaluate how BBB permeability evolves over time from first diagnosis to tumor recurrence using Gallium-68 NOTA-Evans Blue (Ga68-NEB) Positron Emission Tomography/ Magnetic Resonance Imaging (PET/MRI) and Dynamic Contrast Enhanced (DCE)-MRI imaging, and will correlate imaging biomarkers with QPOP-derived therapeutic sensitivities, clinical and radiological response as well as patient outcomes.
Specific Aim 1: To establish primary and corresponding temozolomide/radiotherapy (TMZ/RT)-resistant high grade astrocytic glioma patient-derived organoids.High grade astrocytic glioma organoids can be readily established from primary patient high grade astrocytic glioma tissues either by encapsulating minced high grade astrocytic glioma tissues in matrigel or as free-floating clusters high grade astrocytic glioma tissues in specific organoid medium.Using the established methods, we will generate unique matched pairs of high grade astrocytic glioma organoids with TMZ/RT resistance to model the inevitable tumor recurrence in high grade astrocytic glioma to provide clinically meaningful patient-derived models for subsequent analysis.
Specific Aim 2: To determine the utility of QPOP (Quadratic Phenotypic Optimization Platform)-derived drug combinations in treating recurrent high grade astrocytic glioma.While promising results from ex vivo drug sensitivity using QPOP with patient-derived cells from multiple myeloma and lymphoma has been demonstrated, it has not yet been successfully applied in solid tumors largely due to the challenges in establishing clinically meaningful patient-derived organoids.
Here, using the well-established high grade astrocytic glioma organoid culture systems and the derivation of TMZ/RT-resistant high grade astrocytic glioma organoids, we will determine the drug sensitivity of QPOP-derived drug combinations for high grade astrocytic glioma patients, both in the naïve and TMZ/RT-resistant high grade astrocytic glioma organoids from the same patient.

Specific Aim 3: To evaluate the use of non-invasive imaging tools (Ga68-NEB PET/MRI and DCE-MRI) to track blood brain barrier (BBB) permeability over time in high grade astrocytic glioma patients, and correlate this with pathological biomarkers, therapeutic response determined by follow up standard MRI and clinical outcomes.
The BBB poses significant challenges to the delivery of therapy to brain tumors.This study presents a unique opportunity to examine the relationship between individualized tumoral therapeutic sensitivity, BBB permeability and patient outcomes.We aim to evaluate how BBB permeability evolves over time from first diagnosis to tumor recurrence [assessed Ga68-NEB (Gallium-68 NOTA-Evans Blue) PET/MRI (Positron Emission Tomography/ Magnetic Resonance Imaging), DCE (Dynamic Contrast Enhanced)-MRI imaging] and will correlate imaging biomarkers with QPOP-derived therapeutic sensitivities, clinical and radiological response as well as patient outcomes.Imaging biomarkers evaluated include measurement of standardised uptake values (SUV) of Ga68-NEB uptake, and perfusion parameters (Ktrans, Ve, Vp ) by DEC-MRI in the tumour.
PET imaging with 68 Ga-labeled NOTA (1,4,7-triazacyclononane-N,N′,N″-triacetic acid) conjugated truncated Evans blue (NEB), which complex with serum albumin quickly, and thus most of the radioactivity remain in the blood circulation, allowing for rapid visualization of tracking blood brain barrier (BBB).It has been used clinically to determine blood volume, as a sensitive marker of protein leakage from the vascular lumen in a variety of tissues during inflammation and traumatic injury to evaluate vascular permeability 29 , and as a blood-pool imaging agent 30,31,32 We hypothesize and investigate the utility/performance of 68 Ga-NEB PET/MRI to track BBB permeability as an alternative to DCE-MRI.The challenges of DCE-MRI as a technique for quantitative imaging include technical challenges with obtaining arterial input function (AIF) required to derive the quantitative parameters, sensitivity to motion, and poor repeatability for comparison between scans. 68Ga-NEB PET will specifically identify the boundary and determined the molecular volume of BBB permeability in high grade astrocytic glioma patients.Quantitative analysis with the standardized uptake values (SUV) will be obtained that associated with breakdown of the BBB.These PET quantitative values (SUV) and DCE-MRI derived quantitative values (Ktrans, Ve, Vp) will be evaluated and compared between the imaging modalities and also with response and clinical outcomes.The unique dual-modality imaging PET/MRI offers additional advantages in the evaluation of brain disease.In addition, 68 Ga-NEB will offer unique perspectives for targeted radionuclide therapy after labeling the NEB with therapeutic radioisotopes as a theranostic approach.
The dosage of 68 Ga-NEB will be 185 ± 37 MBq (5 ± 1 mCi).With an injected dose of 185 MBq (5 mCi), the patient would be exposed to an effective radiation dose of 2.94 mSv, which is much lower than the dose limit of 20 mSv for the second risk category defined by the 2007 International Commission on Radiological Protection 33 .
The outcome measures to demonstrate the utility and performance of 68 Ga-NEB PET/MRI for its intended purpose will be 1) the visual analysis of PET/MRI characteristics and pattern; 2) molecular lesion volume measured over the VOIs placed over the "hot" lesions of the involved tissues; 3) the standardized uptake values (SUV) of the lesions using the volume of interest (VOI) method and a unified standard.These parameters will be compared to the standard routine radiological follow up MRI assessments per RANO criteria.
We hypothesize that optimal drug combinations for matched naïve and TMZ/RT-resistant high grade astrocytic glioma organoids will be different and that using drug-screening platform results derived from TMZ/RT-resistant high grade astrocytic glioma organoids to select second-line therapy will be more clinically meaningful and improve outcomes of recurrent high grade astrocytic glioma patients.In the event that the patient undergoes surgery at the time of relapse (approximately 30% of our high grade astrocytic glioma population), organoids will be generated from the second surgical specimen, and the drug sensitivity of the QPOP-derived drug combinations compared with those of treatment naïve and TMZ/RT-resistant organoids from the same patient.

Study Endpoints
The primary endpoint of the study is six-month progression-free survival (PFS 6).PFS is defined as the time from the start of study treatment to documented progression of disease or death; PFS6 refers to the percentage of patients who are alive and free of high grade astrocytic glioma progression at 6 months.This is a common endpoint for clinical trials of second line systemic therapy in high grade astrocytic glioma patients.The PFS6 for recurrent high grade astrocytic glioma ranges from 18-27%. 9

Secondary clinical endpoints:
i. Radiological response assessments at follow up MRI.The determination of radiographic response is as per the Response Assessment in Neuro-Oncology (RANO) criteria. 14ii.Twelve-month overall survival (OS12).OS is defined as the length of time from the start of study treatment, that patients diagnosed with the disease are still alive.OS12 refers to the percentage of patients who are alive at 12 months.

iii. Haematological and non-haematological toxicities. As defined by National Cancer Institute
Common Terminology Criteria for Adverse Events (NCI CTCAE) v5.0

Summary of Study Design
This is an interventional, non-randomized, single site study.In the pilot phase of our study, we will aim to enrol 10 patients who will receive QPOP-guided chemotherapy at the time of first high grade astrocytic glioma recurrence.Subjects will be replaced accordingly if their QPOP analysis is unsuccessful or its results do not lead to therapeutic decision making.

Pre-screening phase:
Patients from NUH and NTFGH (involve in only Pre-screening phase) will be approached for prescreening consent at the time of first suspected diagnosis of a high grade astrocytic glioma and tumor will be harvested at the time of initial surgery/ biopsy for the generation of PDOs.Once the histological diagnosis of high grade astrocytic glioma is confirmed and the patient is planned for adjuvant temozolomide and radiotherapy, our prescreening cohort will undergo baseline standard MRI plus Ga68-NEB PET/MRI and DCE-MRI imaging prior to chemoradiotherapy (Radiological timepoint 1).If patients are admitted to the ward, they will not be sent for the Ga68-NEB PET/MRI and DCE-MRI imaging.These scans should be arranged as outpatient scans only after they are discharged from the ward.In addition, tumor may be harvested for any subsequent procedure that patient might require for brain tumor treatment.Study imaging and further PDO generation will not take place if the patient does not meet the histological criteria or will not be receiving standard adjuvant temozolomide/ radiotherapy.It is estimated that we will need to pre-screen approximately 20 patients in order to achieve our objective of enrolling 10 recurrent high grade glioma patients treated with QPOP-guided systemic therapy.
Patients will then undergo standard-of-care treatment for high grade astrocytic glioma with adjuvant concurrent temozolomide and radiotherapy (TMZ/RT) 1 , and will be evaluated by the primary oncologist routinely with clinical examination, laboratory tests and regular neuro-imaging (performed as standard clinical care and not part of the study).High grade astrocytic glioma organoids will be generated from resected tumour samples and QPOP analyses of the treatmentnaïve high grade astrocytic glioma organoids will be performed (QPOP 1).To model TMZ/RTresistant cells in the setting of recurrent high grade astrocytic glioma, these treatment-naïve organoids will be subjected to chronic exposure of temozolomide, and ionizing radiation.QPOP analyses will be performed on these resistant high grade astrocytic glioma organoids to identify specific drug combinations to guide clinical management at the time of first relapse (QPOP 2).

Main study:
At the time of documented tumor recurrence, eligible patients will be reassessed for suitability to participate in the main study and approached for their informed consent to enter this phase of the study.At this juncture, a small proportion of patients will be deemed suitable for a second operation.
If this is the case, high grade astrocytic glioma organoids will also be generated from the recurrent high grade astrocytic glioma tumour and subject to QPOP analyses (QPOP 3).During the main study, patients will receive QPOP-guided systemic therapy for the treatment of their relapsed high grade glioma and will be assessed regularly for safety and efficacy of this therapy.In addition, patients will undergo standard MRI plus investigational Ga68-NEB PET/MRI and DCE-MRI imaging prior to and 8 weeks (+/-1 week) after QPOP-guided systemic therapy (Radiological timepoints 2 and 3, respectively).If patients are admitted to the ward, they will not be sent for the Ga68-NEB PET/MRI and DCE-MRI imaging.These scans should be arranged as outpatient scans only after they are discharged from the ward.Subsequent to this, radiological assessment of their disease will revert to standard clinical protocols with routine standard MRI imaging.

Choice of dosing regimen
We will have two in vitro drug screening panels.The "clinical care panel" (Panel 1) comprises anticancer agents which have previously been evaluated in recurrent high grade astrocytic glioma: lomustine,temozolomide,vincristine,procarbazine,etoposide,carboplatin,cisplatin,cyclophosphamide,docetaxel,capecitabine, All of these drugs have been determined to have sufficient penetration across the blood-brain barrier and have known adverse event profiles.QPOP will be used to derive top-ranking drug combinations, which will then be validated in vitro.
The "experimental panel" (Panel 2) comprises anti-cancer agents which have promising activity in high grade astrocytic glioma but are not yet routinely used in high grade astrocytic glioma patients: Geftinib, osimertinib, vemurafenib, everolimus, sunitnib, regorafenib, selinexor, marizomib, abemaciclib, ivosidenib, olaparib, metformin.The results of this panel will not be used in the care of our enrolled patients; instead they are hypothesis-generating and intended for us in designing future trials.
The drug combinations generated by QPOP can be divided into 3 categories: 1) combination regimens with published data in the setting of gliomas (category 1); 2) combination regimens where there is published data on intracranial activity and anti-glioma effect of the individual agents either as monotherapy or in combination with other agents, and where there is published safety data on the combination (category 2); and 3) novel drug combinations that have not been assessed for clinical efficacy (category 3).
We will apply the following guidelines for the selection of drug regimen in individual patients: 1.If QPOP analysis results of QPOP 1, 2 and 3 are all available at the time the patient is due to start systemic therapy for the treatment of recurrent high grade astrocytic glioma (i.e., 4-6 weeks after surgery), they will be used in the following order of preference: QPOP 3> QPOP 2> QPOP 1, provided other eligibility criteria for the use of that particular combination's results are met.
2. We will make every effort to use category 1 combinations, but will consider category 2 combinations should the QPOP analysis not generate any category 1 combinations.Decisions to choose category 2 combinations vs defaulting to monotherapy (see point 3) will be discussed with the study team.Doses of drug combinations used should be as close as possible to those used in the published literature.We will not use novel drug combinations (category 3) that have not previously been assessed for clinical safety.
3. If the QPOP combination therapy results do not fall within the criteria for usage, monotherapy may be considered.Temozolomide monotherapy will not be encouraged unless strongly supported by QPOP 2 or 3 results, in which case, a dose schedule different from first-line therapy should be used.
4. Bevacizumab is an FDA approved drug for the treatment of recurrent high grade astrocytic glioma(Avastin (bevacizumab) injection, Genetech, Inc, December 2017), and may be used in combination with lomustine 11 , temozolomide 10 , irinotecan 10 , carboplatin 7 and etoposide 5,7 for the treatment of high grade astrocytic glioma.However, bevacizumab is not included in the drug screening panel because our current PDO models lacks vasculature, hence the functional testing of anti-angiogenic drugs is irrelevant.In order to be relevant to real-world practice, the study will allow the addition of bevacizumab to any of the above agents, provided that investigators adhere as closely as possible to the regimens in the referenced studies.The addition of bevacizumab will not be permitted if the QPOP results suggest an effective combination where there is no published data for the addition of bevacizumab to that particular treatment combination.Deviations from existing data may be permitted only with the agreement of the study team.
The study team will meet to review the QPOP analysis results to agree on the validity of the data and determine the recommended treatment at the time of relapse for each patient on the study.As an additional safety measure, the study team will also conduct a combined review for each patient at the time of their first post-treatment radiologic assessment, to ensure the appropriateness of therapy based on the efficacy and toxicity of the treatment selected.

Safety and efficacy assessments
Each subject will be monitored serially by his/ her neuro-oncologist for the duration of QPOP-guided systemic therapy.This includes a clinical examination and a full blood count, liver function and renal panel prior to each cycle of treatment.NCI CTCAE Version 5 will be used for toxicity and dose modifications.The frequency of visits and dose modifications will take place according to institutional guidelines.Investigational neuro-imaging will be performed by magnetic resonance (MR) imaging, (Ga68-NEB PET/MRI and DCE-MRI), at pre-screening and during the main study, prior to and at 8 weeks (+/-1 week) after QPOP-guided systemic therapy (3 timepoints in total).Imaging on all other occasions will be routine and decided upon by the treating physician.Subjects whose QPOP analysis is unsuccessful or its results do not lead to therapeutic decision making will not be included in the safety and efficacy analysis.

Overall Study Schema
General study schema for QPOP-guided drug combination therapy.Patients will be enrolled to the pre-screening phase of our study at the point of first diagnosis of high grade astrocytic glioma.Established high grade astrocytic glioma PDOs from newly diagnosed patients will be subjected to continuous low dose TMZ and RT as described by the Stupp protocol to model the treatment received in the clinics.QPOP results will be generated from primary high grade astrocytic glioma PDOs (QPOP 1), matched TMZ/RT-resistant high grade astrocytic glioma PDOs (QPOP 2).At the point of diagnosis of high grade astrocytic glioma recurrence, patient will be approached for consent for the main protocol if the PDO generation from their primary tumors was successful.If the patient undergoes surgery at the point of recurrence, tissue will be collected for the generation of PDOs at recurrence (QPOP 3).QPOP-guided second line systemic treatment will be used in order of preference with available QPOP data from QPOP 3> QPOP 2> QPOP 1. PDOs, patient-derived organoids; TMZ, temozolomide; RT, radiation therapy; QPOP, quadratic phenotypic optimization platform.Figure created with BioRender.com.

INVESTIGATOR INFORMATION
The names, titles, and institutions of the investigators are listed in the Contacts for Protocol.If investigators are added after the ethical review board and/or the local regulatory agency have approved the study, these additions will not be considered changes to the protocol, but the Contacts for Protocol will be updated to provide this information.

Study Population and Subject Enrolment
Male and female subjects aged 21 years and above with suspected high grade astrocytic glioma planned for surgery/ biopsy followed by adjuvant chemoradiotherapy will be invited to participate in the pre-screening study.Subjects will only be enrolled in the main study if they had pathologically confirmed high grade astrocytic glioma, and received adjuvant treatment comprising standard-ofcare therapy with surgery/biopsy followed by temozolomide and radiotherapy.In addition, they are required to have had sufficient tumor tissue available for PDO generation at baseline.We aim to to enrol 10 patients who will receive QPOP-guided chemotherapy at the time of first high grade astrocytic glioma recurrence.In order to achieve this, we expect to pre-screen approximately 20 subjects.Subjects will be replaced accordingly if their QPOP analysis is unsuccessful or its results do not lead to therapeutic decision making.These subjects will not be included in our study endpoint analysis.This study is expected to take 18-24 months to complete.
Interim analysis of the PFS6 will be performed after 5 patients have received QPOP-guided chemotherapy.If PFS6 <15% (i.e., none of the patients have achieved 6 months of progression-free survival), this indicates that the trial will likely be futile. 34The study team will then meet to review potential reasons for this and decide if the trial should be discontinued.
Written informed consent for entry into the study will be obtained prior to any study specific procedure.All eligibility criteria and consent forms will be checked before treatment is initiated.

Inclusion Criteria
Subjects may be included in the study only if they meet all of the following criteria: Pre-screening: 1) Patients 21 years of age or older, with ECOG performance status 0 to 2, and with a life expectancy of more than 3 months with suspected high grade astrocytic glioma, fit for treatment comprising standard-of-care therapy with adjuvant temozolomide and radiotherapy if the diagnosis of high grade astrocytoma is pathologically confirmed.2) Signed informed consent obtained before any study specific procedure.Subjects must be able to understand and be willing to sign the written informed consent.* Patients will be enrolled at the time of initial surgery but study imaging and further PDO generation will not take place if the patient is subsequently found not to meet the histological criteria or will not be receiving standard adjuvant temozolomide/ radiotherapy.
All subsequent criteria apply to the main study only: 1) Patients 21 years of age or older, with ECOG performance status 0 to 2, and life expectancy of more than 3 months with pathologically confirmed high grade astrocytic glioma, having undergone first-line standard-of-care therapy with surgery/biopsy followed by temozolomide and radiotherapy.Subjects with truncated adjuvant chemoradiotherapy may be enrolled at the Principal Investigator's discretion.2) Documented tumor progression based on standard clinical, radiological or histological criteria, and deemed suitable for second line systemic therapy.3) Sufficient tumor tissue available for PDO generation at baseline and at least one available or pending QPOP result.4) Adequate organ function as defined by: 1. Bone marrow function i. Haemoglobin ≥ 9g/dl ii.Absolute neutrophil count (ANC) ≥ 1.5 x 10 9 /L iii.Platelet count ≥ 100 x 10 9 /L. 2. Liver function i. Bilirubin < 2.5x upper limit of normal (ULN) ii.Alanine transaminase (ALT) and aspartate transaminase (AST) < 2.5x ULN or < 5x ULN if liver metastases are present iii.Prothrombin time (PT) within the normal range for the institution.3. Renal function i. Plasma creatinine <1.5x institutional ULN 5) Capable of swallowing tablets.6) Recovery from any previous drug-or procedure-related toxicity to National Cancer Institute Common Terminology Criteria for Adverse Events (NCI-CTCAE) version 5.0 Grade 0 or 1 (except alopecia), or to baseline preceding the prior treatment.

Exclusion Criteria (both pre-screening/ main study)
1) Chemotherapy, radiotherapy, surgery, immunotherapy or other therapy within 2 weeks of study entry.2) Pregnancy or breastfeeding at the point where systemic anti-cancer therapy is initiated.
Women of childbearing potential must have a negative pregnancy test at the point where systemic anti-cancer therapy is initiated.Women of childbearing potential and men, must agree to use adequate contraception (barrier method of birth control) while on anti-cancer treatment and until at least 3 months after the last study drug administration.3) Concurrent cancer which is distinct in primary site or histology from the cancer being evaluated in this study EXCEPT cervical carcinoma in situ, treated basal cell carcinoma, superficial bladder tumours (Ta, Tis & T1) or any cancer curatively treated less than 5 years prior to study entry.4) Patients with leptomeningeal dissemination of disease and/or pure spinal high grade gliomas will be excluded.5) Kidney disease which would clinically disqualify the subject from serial MRI scans with gadolinium contrast.6) Previous history of allergy to MRI contrast agents.

Withdrawal Criteria
• Disease progression, • Intercurrent illness that prevents further administration of treatment, • Unacceptable adverse event(s), • Patient becomes pregnant.
• Patient decides to withdraw from the study, or • General or specific changes in the patient's condition render the patient unacceptable for further treatment in the judgment of the investigator.

Subject Replacement
Subjects will be replaced accordingly if: i.Their QPOP analysis is unsuccessful or its results do not lead to therapeutic decision making ii.The subject who withdraws or discontinues from the study prior to the completion of cycle 1 due to reasons other than toxicity or progressive disease.

DOSAGE AND ADMINISTRATION 6.1 Study Treatment
This study does not involve the evaluation of an Investigational Medicinal Product (IMP).All the drugs used in the QPOP drug screening panel have previously been evaluated in high grade astrocytic glioma or other malignancies, with established therapeutic doses.Drug combinations evaluated will be selected only if there is available literature supporting the safety of the combination.Dosages of combination therapy will be in line with published data, unless recommended otherwise by the Study Team.Storage, accountability and prescription of the anti-cancer drugs and their supportive medications will be as per routine clinical use, and in line with institutional guidelines.
Toxicities will be monitored at every cycle and dose modifications may be made at the Investigator's discretion, according to institutional guidelines.There are no specific prohibited medications in this study; investigators should exercise due caution when prescribing concomitant medications while patients are on chemotherapy, in line with standard clinical practice.

Study Assessments
Clinical (within 2 weeks of study enrolment and at the start of each cycle of treatment): • Medical history and physical examination, evaluation of performance status (ECOG) • Adverse event assessment (CTCAE v 5.0) Radiologic (pre-screening phase: prior to chemoradiotherapy; main study: prior to and 8 weeks (+/-1 week)after QPOP-guided systemic therapy): Imaging on all other occasions will be routine and decided upon by the treating physician Laboratory (within 2 weeks of study enrolment and at the start of each cycle of treatment, +/-3 days window period): • Laboratory parameters including haematology (FBC), chemistries, others as clinically indicated.
Final Study Visit: Clinical assessment within 30 days from the time of study discontinuation/completion of study treatment.

Post-Study follow-up:
To obtain meaningful data on survival outcomes, assessments of disease and survival status will be made at regular intervals after patients discontinue from study therapy.Assessments will continue until documented disease progression or death whichever occurs first.
During the post-study follow-up period, information will be collected regarding date of disease progression or death.The study will be closed when, in the opinion of the principal investigator, sufficient data have been obtained for completion of the final study manuscript.

Safety
After a patient discontinues study therapy, the investigator should make every effort to continue to evaluate the patient for delayed toxicity by clinical and laboratory evaluations as clinically indicated.The patient must be followed approximately every 30 days until toxicity resolves.

SPECIAL TESTS
All subjects must have at least one tumour lesion that is suitable for biopsy or surgical resection at baseline.
The protocol includes ancillary studies on machine-learning guided radiomics and tumour histopathology, and will be carried out for patients who have given specific consent for these studies.Standard MRI brain with or without perfusion sequences are typically performed at baseline and after therapy in order to evaluate radiological response.In this study, we plan to add Ga68-NEB (Gallium-68 NOTA-Evans Blue) PET imaging and DCE-MRI imaging to examine BBB permeability and assess in-vivo drug effects on tumor vasculature over time.These studies are planned at 3 timepoints: 1) During the pre-screening phase, following confirmation of histological diagnosis and prior to chemoradiotherapy and 2) during the main study, prior to and 8 weeks (+/-1 week) after QPOP-guided systemic therapy.Radiological biomarkers will be correlated with radiological response based on follow up with routine MRI and clinical outcomes as well as therapeutic sensitivities.

Radiomics
MRIs obtained at initial diagnosis and subsequent scans will be subjected to radiomic feature extraction, and correlated with spatially annotated histopathological biopsies opportunistically obtained as part of standard-of-care surgical resection both at initial diagnosis (chemotherapy-and radiotherapy-naïve) and at recurrence.Annotation of biopsy site would be done on T1-stereotaxy and pathology images would be correlated with spatial radiomic features and clustering.In addition, part of the biopsy will be used to generate high grade astrocytic glioma organoids, and tested for chemosensitivity towards both temozolomide and the optimal drug combination the participant will be treated with.These spatial properties of the high grade astrocytic glioma biopsies will be used for model building for prediction of post-chemotherapy recurrence.

b) Histopathology
Paraffin-embedded blocks of high grade astrocytic glioma tumour will be stained to assess DNA damage response (ɣH2AX, RAD51), growth factor receptors (EGFR, PDGFR) and hypoxia markers (HIF1A) in relation to blood vessels and axons.Molecular analyses of TP53, ATRX, IDH1, IDH2, BRAF, and EGFRvIII mutations, MGMT methylation status, and 1p19q co-deletion would be performed as part of routine care.

c) Plasma biomarkers
Approximately Fifteen to Eighteen millilitres of blood will be collected together with routine blood tests for plasma biomarkers including circulating tumor DNA, proteomics and DNA methylation at these time points.These will be stored in the NUH tissue repository for future analysis.
• Prescreening -At first MRI scan before chemoradiotherapy • Main study -At baseline and 8-week post QPOP-guided chemotherapy

Definitions Adverse Event
An adverse event is defined as any untoward medical occurrence in a subject administered a pharmaceutical product and which does not necessarily have a causal relationship with the treatment.An adverse event can therefore be any unfavorable and unintended sign (including an abnormal laboratory finding), symptom, or disease temporally associated with the use of a medicinal (investigational) product, whether or not the event is considered causally related to the use of the product.Such an event can result from use of the drug as stipulated in the protocol or labelling, as well as from accidental or intentional overdose, drug abuse, or drug withdrawal.Any worsening of a pre-existing condition or illness is considered an adverse event (with the exception of cancer).Clinical signs and symptoms of disease progression are considered adverse events.Laboratory abnormalities and changes in vital signs are considered to be adverse events only if they result in discontinuation from the study, necessitate therapeutic medical intervention, meet protocol specific criteria and/or if the investigator considers them to be adverse events.

CTCAE term (adverse event description) and grade:
The descriptions and grading scales found in the revised NCI Common Terminology Criteria for Adverse Events (CTCAE) version 5.0 will be utilized for adverse event reporting.A copy of the CTCAE version 5.0 can be downloaded from the CTEP web site (http://ctep.cancer.gov/reporting/ctc.html).
• Attribution of the adverse event: -Definite -The adverse event is clearly related to the study treatment.
-Probable -The adverse event is likely related to the study treatment.
-Possible -The adverse event may be related to the study treatment.
-Unlikely -The adverse event is doubtfully related to the study treatment.
-Unrelated -The adverse event is clearly NOT related to the study treatment.

Serious Adverse Event
A serious adverse event is any untoward medical occurrence at any dose that meets any of the following criteria, whether related to study drug or not: Death of Subject-An event that results in the death of a subject.Life-Threatening-An event that, in the opinion of the investigator, would have resulted in immediate fatality if medical intervention had not been taken.This does not include an event that would have been fatal if it had occurred in a more severe form.
Hospitalization-An event that results in an admission to the hospital for any length of time.This does not include an emergency room visit or admission to an out-patient facility.Prolongation of Hospitalization-An event that occurs while the study subject is hospitalized and prolongs the subject's hospital stay.Congenital Anomaly-An anomaly detected at or after birth, or any anomaly that results in fetal loss.
Persistent or Significant Disability/Incapacity-An event that results in a condition that substantially interferes with the activities of daily living of a study subject.Disability is not intended to include experiences of relatively minor medical significance such as headache, nausea, vomiting, diarrhoea, influenza, and accidental trauma.

Important Medical Event Requiring Medical or Surgical Intervention to Prevent
Serious Outcome An important medical event that may not be immediately life-threatening or result in death or hospitalization, but based on medical judgment may jeopardize the subject and may require medical or surgical intervention to prevent any of the outcomes listed above (i.e., death of subject, life-threatening, hospitalization, prolongation of hospitalization, congenital anomaly, or persistent or significant disability/incapacity).
Examples of such events include allergic bronchospasm requiring intensive treatment in an emergency room or at home, blood dyscrasias or convulsions that do not result in inpatient hospitalization, or the development of drug dependency or drug abuse.Spontaneous Abortion Miscarriage experienced by study subject.
Elective Abortion Elective abortion performed on study subject.

Collecting, Recording and Reporting of "Unanticipated Problems Involving Risk to
Subjects or Others" -UPIRTSO events to the NHG Domain Specific Review Boards (DSRB) UPIRTSO events refers to problems, in general, to include any incident, experience, or outcome (including adverse events) that meets ALL of the following criteria: 1. Unexpected In terms of nature, severity or frequency of the problem as described in the study documentation (eg: Protocol, Consent documents etc).

Related or possibly related to participation in the research
Possibly related means there is a reasonable possibility that the problem may have been caused by the procedures involved in the research; and

Risk of harm
Suggests that the research places participants or others at a greater risk of harm (including physical, psychological, economic, or social harm) than was previously known or recognized.
Reporting Timelines for UPIRTSO Events to the NHG DSRB: 1. Urgent Reporting: All problems involving local deaths, whether related or not, should be reported immediatelywithin 24 hours after first knowledge by the investigator.

Expedited Reporting:
All other problems must be reported as soon as possible but not later than 7 calendar days after first knowledge by the investigator.

Safety Monitoring Plan
The study team will meet to review the QPOP analysis results, agree on the validity of the data and determine the recommended treatment at the time of relapse for each patient enrolled onto the study.The investigator is responsible for appropriate medical care of subjects during the study.Investigator will review all patients before each treatment cycle for monitoring of toxicity.If a subject reports an adverse event, the investigator should follow the subject until the event is either resolved or assessed as stable.Safety data will be monitored every week by reviewing the toxicity data by the principal investigator, at least one other investigator and the research nurse.All SAE will be reviewed.
Collection period of SAE is from first QPOP-guided study dosing until 28 days after discontinuation.
As an additional safety measure, the study team will also conduct a combined review for each patient at the time of their first post-treatment radiologic assessment, to ensure the appropriateness of therapy based on the efficacy and toxicity of the treatment selected.Interim analysis of the PFS6 will be performed after 5 patients have received QPOP-guided chemotherapy.If PFS6 <15% (i.e., none of the patients have achieved 6 months of progression-free survival), this indicates that the trial will likely be futile. 34The study team will then meet to review potential reasons for this and decide if the trial should be discontinued.

DATA ANALYSIS 10.1 Data Quality Assurance
Although a research nurse or study coordinator may interview subjects and complete the case report forms, the investigator will verify that all data entries are accurate and correct, including verification that the subject fulfils the criteria for entry into the study before study medication is dispensed.Physical examinations have to be performed by a registered medical practitioner.All relevant forms including End of Treatment Forms must be completed for each patient upon completion or withdrawal from the study.

Data Entry and Storage
All data obtained in the study described in this protocol will be recorded on electronic case report forms (eCRFs) named REDCAP.The eCRFs will be completed chronologically and updated regularly in order to reflect the most recent data on the patient included in the study.
The investigators of the trial will be responsible for the integrity and accuracy of the data collected.
All electronic data will be kept in system and only authorised study team personnel with Redcap access can access the data.Prior to the start of the study, the Principal Investigator will delegate personnel who are authorized to make or change entries on the eCRFs.
Each eCRF pages must be completely filled by referring to study source documents.All the eCRF pages which include, but not limited to the registration form, the treatment forms and the follow-up status form must be reviewed by authorized investigator throughout the study conduct.
Although a study co-ordinator may interview subjects, the investigator must verify that all data entries are accurate and correct, including verification that the subject fulfils the criteria for entry into the study.Physical examinations have to be performed by a registered medical practitioner.
The End of Treatment Form must be completed for each patient upon completion or withdrawal from the study.

SAMPLE SIZE AND STATISTICAL METHODS
This is an interventional, non-randomized, single site study.The primary purpose of this study is not hypothesis testing, but to assess the feasibility of QPOP-guided therapy for recurrent high grade astrocytic glioma to be used in a larger scale study.Therefore, this study does not have a formal sample size, but rather, a set benchmarks to determine feasibility.We plan to enrol a total of 10 patients who receive QPOP-guided chemotherapy at the time of first high grade astrocytic glioma recurrence.We expect this to be sufficient for determining the feasibility of a phase II trial in which there will be formal sample size calculations for the achievement of PFS6 > 30%. 35In addition, a phase II study will only be considered feasible if there is successful organoid generation and QPOP analysis in > 50% of samples collected.In this study, subjects will be replaced accordingly if their QPOP analysis is unsuccessful or its results do not lead to therapeutic decision making.

Informed Consent
Obtaining and documenting informed consent will be performed by the Investigator or his/her representative in compliance with the ICH GCP guidelines and the ethical principles that have their origin in the Declaration of Helsinki.Prior to the initiation of any screening or study-specific procedures, the investigator or his/her representative will explain the nature of the study to the subject and answer all questions regarding this study.The patient will be told of his or her right to withdraw from the study at any time without any penalty with regards to the continuation of care at this institution and by the same physicians chosen by him.Each informed consent will be reviewed, signed and dated by the subject and the person who administered the informed consent.A copy of each informed consent will be given to the subject and each original will be kept in the Investigator Site File.

IRB Review
Each participating institution must provide for the review and approval of this protocol and the associated informed consent documents by the IRB / NHG DSRB.
Good Clinical Practice (GCP) requires that the clinical protocol, the Investigator's Brochure, the informed consent and all other forms of subject information related to the study (e.g., advertisements used to recruit subjects) and any other necessary documents be reviewed by an IRB / NHG DSRB.IRB / NHG DSRB approval of the protocol, informed consent and subject information and/or advertising, as relevant, will be obtained prior to study commencement.Any amendments to the protocol will require IRB / NHG DSRB approval prior to implementation of any changes made to the study design.During the conduct of the study, the investigator will promptly provide written reports (e.g., ICH Expedited Reports or any additional reports required by local regulations) to IRB / NHG DSRB of any changes that affect the conduct of the study and/or increase the risk to subjects.

Confidentiality of Data and Patient Records
Information collected for this study will be kept confidential.This confidentiality is extended to cover testing of biological samples and genetic tests in addition to the clinical information relating to the participating subjects.Patient records, to the extent of the applicable laws and regulations, will systemic therapy (Radiological timpepoints 2 and 3) for evaluation of tumor response.Subsequent to this, radiological assessment will revert to standard clinical protocols with routine standard MRI imaging.d.Applicable only for female participants or women of childbearing potential.e. Prior to chemoradiotherapy f.
If surgery is deemed to be too risky for patient at pre-screening phase, patient biopsy sample will be used instead if there is sufficient tumor tissue.In addition, tumor may be harvested for any subsequent procedure that patient might require for brain tumor treatment.g.If there is a second tissue collection at relapse.